This disclosure is concerned generally with processes for detecting the presence of Neisseria bacteria utilizing labelled antibodies.
The importance of being able to quickly and accurately determine the presence of Neisseria bacteria, particularly Neisseria gonorrhoeae, is well-appreciated. Conventional tests for detecting the presence of organisms such as N. gonorrhoeae require the preparation of bacteria cultures or the use of serological methods. Such tests, however, have well-recognized limitations. See, for example, the publication "International Symposium on Gonorrhea", B. B. Diena, Ed., a collection of papers presented at the October, 1973 International Symposium on Gonorrhea sponsored by the Health Protection Branch, Health and Welfare Canada, Ottawa, especially at page 34 et seq.
A relatively simple and quick enzymatic test for the presence of Neisseria bacteria is disclosed in the related patent application cited above entitled "Detecting Neisseria Bacteria". That test is based upon the discovery of an enzyme in Neisseria bacteria which is released during lysis thereof which appears to be specific to Neisseria bacteria. Although the full structure and composition of the enzyme has not been explained and no identification therefor has been found in the literature, the enzyme has the capability for oxidizing 1,2-propanediol and reducing nicotinamide-adenine-dinucleotide (NAD) to NADH. In view of those property characteristics, the name 1,2-propanediol dehydrogenase has been suggested for the enzyme, and that appellation will be used throughout this specification.
In another related application cited above entitled "Detection of Neisseria Bacteria by Immunoassay" is disclosed the use of antibodies to inhibit the activity of the enzyme 1,2-propanediol dehydrogenase. And in yet another application cited above entitled "Immunoassay of Neisseria Bacteria Via (NH.sub.4).sub.2 SO.sub.4 Precipitation" is disclosed a modification of that method wherein (NH.sub.4).sub.2 SO.sub.4 precipitation of the antigen-antibody complex acts to concentrate the enzyme, remove interfering materials, and thereby improve the speed and precision of the assay.
Immunological reactions are recognized as being highly specific biochemical reactions wherein a first protein, known as the antigen, bonds with a protein specific to the antigen, termed on antibody, to form an immunologically complexed protein. In the vast majority of instances, the reaction will occur even when the antigen is modified through the addition of a radioactive label or tracer. The antibody-antigen complexes formed thereby are radioactive and can be separated from uncomplexed reactants by various well-established means. Inasmuch as the measurement of radioactivity is a known and sensitive procedure, a quantitative assay is possible for any protein to which a specific antibody is available.
Two closely related assay methods utilizing radiolabelled antibodies to convert an unknown soluble antigen into a radioactive product are discussed in "Handbook of Radioimmunoassay", edited by Guy E. Abraham, Vol. 5, Chap. 4, pages 131-177, Marcel Dekker, Inc., New York and Basel (1977).
The first method, termed immunoradiometric assay (IRMA), contemplates the following general procedure. The unknown antigen is first reacted with soluble purified radiolabelled antibodies. The radiolabelled complex remains in solution while unused radiolabelled antibodies are removed via a second reaction with a solid phase antigen or antigen immunoadsorbent. The amount of radioactivity remaining in solution is a direct function of the antigen concentration.
The second method, variously termed "two-site IRMA", "junction test", and "sandwich technique", contemplates first the insolubilization of the unknown antigen by reaction with solid phase antibodies, and then carrying out the reaction with soluble labelled antibody. In this practice, the labelled complex is insoluble and unreacted labelled antibody can be washed away. An increase in the amount of unknown antigen results in an increase in radioactivity in the solid phase. The use of this second method is limited to antigens which can either (1) bind simultaneously to at least two antibodies, or (2) bind to a radiolabelled antibody after some nonimmunological insolubilization procedure.
Thus, any particular proteinaceous material will consist of various entities, e.g., protein molecules, cells, etc., which do not adhere to each other. Accordingly, proteinaceous matter deposits as a single layer when brought into contact with a substrate. And no other arbitrary protein will adhere to the layer of deposited protein. Nevertheless, a protein which will specifically react to the protein adsorbed onto or chemically bonded, e.g., covalently bonded, to the substrate will immunologically bond thereto. This phenomenon has given rise to immunoassay methods utilizing the "sandwich technique" whereby a layered structure is formed on a base substrate.
Those methods can be generally described as comprising the following steps. A substrate is prepared having a first layer of a protein physically adsorbed or covalently bonded thereon which can be utilized to test suspected solutions for the presence of a protein specifically reactive to the adsorbed or covalently bonded protein. The substrate having the adsorbed protein is then contacted with a medium containing the substance to be analyzed, this substance including particles which bond to the proteinaceous matter adsorbed on the substrate. Finally, the two-layer composite is contacted with a medium containing labelled or tagged antibodies to the particles of the second layer. Customarily, the label or tag will consists of a radioactive isotope or a fluorescent group from which emanations occur. Such a unit can be chemically integrated into an entity which, because of its presence in extremely dilute concentrations, is itself exceedingly difficult to detect, and which, because of the emanations arising therefrom, can be readily detected even in very small quantities. Therefore, detection of the labelling or tagging unit simultaneously confirms the presence of the entity into which it is integrated.
In summary, a composite or sandwich is produced comprising a first or inner layer containing unlabelled antibodies immobilized on a substrate; a second or intermediate layer containing antigens which are bonded to the immobilized antibodies; and a third or outer layer having labelled antibodies which are bonded to the antigens of the intermediate layer. The surface of the composite is monitored for the presence of the labelling or tagging units.
The general steps contemplated in IRMA are set out below wherein Ag refers to antigen and Ab* designates purified radiolabelled antibodies: EQU (1) Ag+Ab*.fwdarw.AgAb*+Ab* ##EQU1##
The general steps involved in two-site IRMA or the sandwich technique are reported below wherein Ab refers to antibody, Ag designates antigen, and Ab* describes purified radiolabelled antibodies: EQU (3) solid phase Ab+Ag.fwdarw.insolubilized Ag EQU (4) insolubilized Ag+Ab*.fwdarw.insolubilized AgAb*+Ab* EQU (5) insolubilized AgAb*+Ab* .sup.wash remove Ab*
It will be appreciated that these two methods differ from conventional radioimmunoassay (RIA) in that the antigen to be measured is assayed directly by reaction with excess radiolabelled antibodies, rather than by competition with a labelled antigen derivative for a limited amount of antibody. The related case cited above entitled "Detection and Quantitation of Neisseria by the Radioimmunoassay of an Enzyme Present in Neisseria Bacteria" is illustrative of a typical radioimmunoassay method. Thus, the IRMA and RIA techniques are compared below: ##EQU2## The asterisk denotes radiolabelling.
In general, the sandwich technique provides better sensitivity, i.e., the minimal detectable quantity is lower, than IRMA. Likewise, the precision and accuracy of the sandwich technique are generally superior to those of IRMA. Consequently, those advantages have led to the former method of assay to be preferred and the working examples provided hereinbelow are directed to the two-site IRMA or sandwich technique.